The ocular ciliary epithelium is the site of aqueous humor secretion in the mammalian eye. The ciliary epithelium is also the main target tissue to reduce intraocular pressure in the treatment of glaucoma. The ionic composition of aqueous humor indicates that the major anion transported by the ciliary epithelium is chloride (Cl-) via Cl- channels. The activation of these Cl- channels have been observed by swelling nonpigmented ciliary epithelial cells, in vivo and in vitro, under anisosmotic conditions, and they are rate-determining the aqueous humor secretion during volume regulation. Two candidate genes had been identified to participate in volume regulation via chloride conductance in nonpigmented ciliary epithelial cells: a) the human homologue of pICln, a Cl- channel regulator, and b) the human homologous to the non-synaptic Cl-channel ClC-3. In this research grant the PI proposes that pICln, is the Cl-channel regulator of ClC-3 channels in NPE cells. The main objective of the proposed project is to analyze the relationship between structure and function of pICln, and ClC-3. To achieve this goal we intend the following: 1) The identification and characterization of unique domains of primary sequence in pICln (i.e. nucleotide binding site, Ca2+ binding sites, acidic domains, phosphorylation sites) and ClC-3 (i.e., phosphorylation sites for PKC, and Ca2+/calmodulin-dependent protein kinase II), to gain insight into the mechanisms underlying pICln and ClC-3 biogenesis, subunit assembly and interaction with other proteins. The generation of polyclonal antibodies to purified pICln and ClC-3 proteins expressed in bacteria and against synthetic peptides with help to elucidate their structural-functional properties. 2) Application of molecular strategies such as site specific mutagenesis in unique binding sites or phosphorylation sites residues of pICln and ClC-3 will permit to study these functional domains in response to hypotonic swelling stimuli. This type of approach, coupled with antisense strategy will help to gain insight into the mechanisms underlying Cl-transport in the ciliary epithelium. Overexpression of pICln or ClC-3 in cells deficient in these channels also should permit to determine whether the kinetic behavior and current-voltage relationship correspond to the specific Cl-channel. 3) To determine whether there is differential gene expression of pICln and ClC-3 along the distinct regional areas or segments of the human ciliary epithelium, namely the pars plicata and pars plana, by applying reverse transcript PCR. The goal of these studies will be to obtain basic information about pICln and ClC-3 gene expression, distribution of NPE and PE cells and whether anisosmotic treatments can lead to regulation of pICln and ClC-3 at the transcriptional and/or posttranslational level. 4) To determine the organization of the human pICln and ClC-3 genes at their 5' flanking region. The goal in these studies will be to obtain information from genomic clones of the putative regulatory elements present in these genes.
